A system for efficient assembly of HCV structural proteins into HCV-like particles (HCV-LPs) in insect cells has been developed in our laboratory. These noninfectious HCV-like particles have similar morphologic, serologic and biophysical properties as the putative virions isolated from HCV infected humans. In contrast to recombinant subunit vaccines, the viral proteins of HCV-like particles may be presented in a native, virion-like conformation and may therefore be superior in eliciting a protective humoral and cellular immune response. The humoral and cellular immunogenicity of the HCV-LP had previously been demonstrated in the mouse and baboon models. In addition, we demonstrated the immunogenicity and induction of protective immunity by HCV-LP in chimpanzees. Chimpanzees, two in each group, were immunized with HCV-LP or HCV-LP adjuvant ASO1B. After four immunizations over an eight-month period, all animals developed strong HCV-specific cellular immune response including IFN-gamma and IL-2, CD4 and CD8 T-cell and proliferative lymphocyte responses against core, E1 and E2. The chimpanzees in both groups were challenged with a 100 CID50 of HCV CG1B inoculum. Upon challenge with HCV, one chimpanzee developed transient viremia with low HCV RNA titers (10E3-4 copiesml) in the third and fourth weeks post-challenge. The three other chimpanzees became infected with higher levels of viremia (10E4-5 copiesml) but their viral levels became unquantifiable (< 1000 copiesml) 10 weeks post-challenge. After HCV challenge, all four chimpanzees demonstrated a significant increase in peripheral IFN-gamma T-cell and proliferative responses as well as the presence of intrahepatic T-cell response against the HCV structural proteins. These T-cell responses coincided with the fall in HCV RNA level. In comparison, four nave chimpanzees were infected with the same HCV inoculum, and three developed persistent infection with viremia in the range of 10E5-6 copiesml. Our study suggests that HCV-LP immunization induces strong HCV-specific cellular immune responses and confers partial protection against HCV challenge in the chimpanzee model. In an effort to improve and broaden the immunogenicity of HCV-LP, we have engineered T cell epitopes from the nonstructural genes into the HCV-LP. Our preliminary data showed that T cell response against the nonstructural epitopes carried by the chimeric HCV-LP were induced in mice. In addition, we are combining The HCV-LP approach with other modalities of immunization, such as plasmid DNA, in a prime-boost regimen.[unreadable] [unreadable] Understanding the structural features of HCV is crucial in designing immunogen that would induce protective immunity. The structural details of hepatitis C virus (HCV) have been elusive because of the lack of a robust tissue culture system for producing an adequate amount of virions from infectious sources for in-depth three-dimensional (3D) structural analysis. Using both negative-stain and cryo-electron microscopy (cryoEM), we show that HCV virions isolated from cell culture have a rather uniform size of 50 nm in diameter and that recombinantly expressed HCV-like particles (HCV-LPs) have similar morphologic, biophysical, and antigenic features. 3D reconstructions were obtained from HCV-LPs having the same size as the HCV virions in the presence and absence of monoclonal antibodies bound to the E1 glycoprotein. The 3D reconstruction of HCV-LP reveals a multilayered architecture, with smooth outer-layer densities arranged in a fishbone configuration. Reconstruction of the particles in complex with anti-E1 antibodies shows that sites of the E1 epitope are exposed and surround the 5-, 3-, and 2-fold axes. The binding pattern of the anti-E1 antibody and the fitting of the structure of the dengue virus E glycoprotein into our 3D reconstructions further suggest that the HCV-LP E1 and E2 proteins form a tetramer (or dimer of heterodimers) that corresponds morphologically and functionally to the flavivirus E homodimer. This first 3D structural analysis of HCV particles offers important insights into the elusive mechanisms of HCV assembly and maturation.